Improved lipid production and component of mycosporine-like amino acids by co-overexpression of amt1 and aroB genes in Synechocystis sp. PCC6803

Implementing homologous overexpression of the amt1 (A) and aroB (B) genes involved in ammonium transporter and the synthesis of mycosporine-like amino acids (MAAs) and aromatic amino acids, respectively, we created three engineered Synechocystis sp. PCC6803 strains, including Ox-A, Ox-B, and Ox-AB, to study the utilization of carbon and nitrogen in cyanobacteria for the production of valuable products. With respect to amt1 overexpression, the Ox-A and Ox-AB strains had a greater growth rate under (NH4)2SO4 supplemented condition. Both the higher level of intracellular accumulation of lipids in Ox-A and Ox-AB as well as the increased secretion of free fatty acids from the Ox-A strain were impacted by the late-log phase of cell growth. It is noteworthy that among all strains, the Ox-B strain undoubtedly spotted a substantial accumulation of glycogen as a consequence of aroB overexpression. Additionally, the ammonium condition drove the potent antioxidant activity in Ox strains with a late-log phase, particularly in the Ox-B and Ox-AB strains. This was probably related to the altered MAA component inside the cells. The higher proportion of P4-fraction was induced by the ammonium condition in both Ox-B and Ox-AB, while the noted increase of the P1 component was found in the Ox-A strain.

The outlined connection between the metabolism of carbon and nitrogen in cyanobacteria is a well-known process 1,16,17 .Through the sequential action of two enzymes, including glutamine synthetase (GS) and glutamate synthase (GOGAT), in the glutamate/glutamine cycle, ammonium is directly transferred from the media via the Amt transporter and integrated into carbon skeletons 10,16 .The amidation of glutamate to glutamine is catalyzed by the GS enzyme, and the GOGAT enzyme catalyzes the reductive transfer of the amide group from glutamine to 2-oxoglutarate (2-OG) 18 .Despite the fact that cyanobacteria lack 2-oxoglutarate dehydrogenase, it was found that 2-oxoglutarate decarboxylase (OGDC) and succinic semialdehyde dehydrogenase (SSA-DH) work together to fill the gap between 2-OG and succinate in the TCA cycle 19 .The alternative route to succinate is the γ-aminobutyrate (GABA) shunt from glutamate which is composed of glutamate decarboxylase (GDC), GABA aminotransferase (GABA-AT), and SSA-DH 20,21 .A malic enzyme (ME) converts malate to pyruvate, which releases CO 2 , and proceeds to flow in a number of other routes, including the formation of acetyl-CoA, phosphoenolphyruvate (PEP), carotenoids, and isoprene molecules.In one hand, main acetyl-CoA precursor could flow to many main pathways, including TCA cycle, fatty acid and lipid syntheses (FAS II), and polyhydroxybutyrate (PHB) production [22][23][24][25][26] .In cyanobacteria, membrane lipids can be hydrolyzed by lipase A enzyme, which is encoded by lipA gene.The thereby produced free fatty acids (FFAs) can then be recycled into fatty acyl-ACP by the acyl-ACP synthetase, encoded by aas gene 1,2,27,28 , or they can be secreted outside of the cell as extracellular FFAs.On the other direction, pyruvate can be converted to phosphoenolpyruvate (PEP) by phosphoenolpyruvate synthase (PPS).To synthesize aromatic amino acids and mycosporine-like amino acids (MAAs), PEP combines with erythrose-4-phosphate (E4P) to generate 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) which can be converted to 3-dehydroquinate (DHQ) by the 3-dehydroquinate synthase enzyme, encoded by aroB (slr2130).The DHQ is an important precursor for aromatic amino acid syntheses, and 4-deoxygadusol (4-DG) which is the substrate for the syntheses of MAA and MAA derivatives 29 .To date, over 40 different derivative MAAs and MAAs, such as shinorine, porphyra-334, palythine, and mycosporine-2-glycine, have been identified 30,31 and are well-known as photoprotectants or neutrally cellular sunscreens due to their chemical structures 32 .MAAs might be considered as an intracellular nitrogen reserve, although the processes regulating their breakdown are currently unclear 33,34 .
In this study, we constructed three mutant strains, including Synechocystis sp.PCC 6803 overexpressing the amt1 (or sll0108) gene encoding NH 4 + permease involved in ammonium transporter (Ox-A strain), Synechocystis sp.PCC 6803 overexpressing the aroB (or slr2130) gene encoding 3-dehydroquinate (DHQ) synthase involved in mycosporine-like amino acid (MAA) and aromatic amino acid syntheses (Ox-B), and a double overexpression of the Ox-AB strain.With a system of (NH 4 ) 2 SO 4 supplementation, we discovered a substantial flow between nitrogen and carbon metabolism in these modified strains.All Ox strains had elevated lipid levels, and various components of MAAs were also present.

Engineered Synechocystis sp. PCC6803 strains
Three constructed Synechocystis sp.PCC6803 strains, including Ox-A, Ox-B, and Ox-AB (Table 1 and Fig. 1) were obtained by single or double homologous recombination.First, the recombinant plasmids (Table 1), including pECm_amt1, pECm_aroB, and pECm_amt1/aroB, were generated by ligating each amplified gene fragment with pEERM vector.For single and double recombinant plasmids, native amt1, aroB, and amt1_aroB gene fragments were separately ligated between flanking regions of the psbA2 gene of the pEERM vector and the upstream region of Cm r cassette (Table 1).For PCR analysis using UUSpsbA2 and DDSpsbA2 as a pair of primers (Supplementary Information Table S1), the positive colonies of Ox-A were clones no. 3 and no. 4 as shown by the expected size of about 4.0 Kb in Lanes 4 and 5, respectively (Fig. 1B).For Ox-B, the PCR product using UUSpsbA2 and DDSpsbA2 primers was found in Clones No. 3-5, as shown in Lanes 4-6 with the fragment size of about 3.9 Kb (Fig. 1C).Those two single gene overexpressing strains were ultimately confirmed by double homologous recombination as expected.For a double overexpression of amt1 and aroB genes in Synechocystis WT (Ox-AB), only  S1), with the expected sizes of about 4.0, 1.8, 3.5, and 3.2 Kb, respectively, were correctly confirmed in Lanes 2, 4, 6, and 8, respectively, compared with the negative control of each specific pair of primers in Lanes 1, 3, 5, and 7, respectively (Fig. 1D).The higher transcript levels of gene overexpression, including amt1 of Ox-A, aroB of Ox-B, and both of them in Ox-AB, were verified by RT-PCR (Fig. 2A).

Growth, intracellular pigments, and oxygen evolution rate of engineered strains
All strains were cultured for 16 days in both regular BG 11 medium and with 8.8 mM (NH 4 ) 2 SO 4 supplementation in BG 11 medium without NaNO 3 (BG 110 + (NH 4 ) 2 SO 4 ) (Figs. 3 and 4).It is important to note that the growth of all strains in BG 110 + (NH 4 ) 2 SO 4 medium was lower than it was in normal BG 11 .However, the amt1-overexpressing strain (Ox-A) showed a higher growth rate than other strains under both growth conditions (Fig. 3A,B).As expected, Ox-A and Ox-AB strains with overexpressing amt1 gene in ammonium transporter had higher growth than WT and Ox-B strains under (NH 4 ) 2 SO 4 -supplemented condition, as well as higher growth rates (Fig. 3B).These were in line with the first four days of cultivation, when cell cultured flasks of the Ox-A and Ox-AB strains contained deeper green than those of the WT and Ox-B strains (Fig. 4).On the other hand, the constant  S1) were used to confirm the complete integration of all transformants by PCR analysis.For the double homologous recombination (A), the amt1 or aroB gene recombination occurred between the conserved sequences of psbA2 gene in Synechocystis sp.PCC 6803 wild type (WT).For Ox-A strain (B), PCR products using UUSpsbA2 and DDSpsbA2 primers, Lane M: GeneRuler DNA ladder (Fermentas Life Sciences, MD, USA); Lane 1: negative control using WT as template, Lanes 2-6: Clones No. 1-5.Only positive clones no. 3 and 4 in respective Lanes 4 and 5, were obtained.For Ox-B strain (C), PCR products using UUSpsbA2 and DDSpsbA2 primers, Lane M: GeneRuler DNA ladder, Lane 1: negative control using WT as template; Lanes 2-6: Clones No. 1-5.Only positive Clones No. 3-5 in respective Lanes 4-6, were obtained.For Ox-AB strain (D), the single homologous recombination was confirmed by PCR using various pairs of primers (Supplementary Information Table S1), Lane M: GeneRuler DNA ladder; Lanes 1 and 2: negative control using WT as template and a transformant, respectively, with UUSll0108_F and Cm_R primers; Lanes 3 and 4: negative control using WT as template and a transformant, respectively, with Cm_F and pEbb_R primers, Lanes 5 and 6: negative control using WT as template and a transformant, respectively, with pEbb_F and Slr2130_R primers, Lanes 7 and 8: negative control using WT as template and a transformant, respectively, with Slr2130_F and pEbb_R primers.
accumulation of chlorophyll a contents occurred in all strains under both growth conditions, except for the Ox-B under the (NH 4 ) 2 SO 4 -supplemented condition, which had the lowest amounts among all strains with light green culture during the first four days of cultivation (Figs.3C,D, and 4).It was found that all strains noted an enhanced accumulation of carotenoids during the duration of 16 days of growth in normal BG 11 medium, in particular the WT strain (Fig. 3E).In exception, the Ox-B strain also contained the lowest amount of carotenoids under (NH 4 ) 2 SO 4 -supplemented condition among all strains during the first 8 days of growth (Fig. 3F).Additionally, the oxygen evolution rates of all strains were increased during late-log phase of growth, and subsequently decreased during early-stationary phase (Fig. 3G,H).It is worth noting that the Ox-A and Ox-B strains possessed a lower oxygen evolution rate than WT, while Ox-AB has a higher oxygen evolution rate under normal BG 11 condition regarding its less chlorophyll a content (Fig. 3G).The reduction in oxygen evolution rate of the WT and Ox-AB strains was influenced by the BG 110 + (NH 4 ) 2 SO 4 condition (Fig. 3H).

Productions of intracellular lipids, extracellular free fatty acids (FFAs), glycogen, and PHB under ammonium sulfate supplementation
We found increased levels of intracellular lipids in Ox strains, especially Ox-A and Ox-AB strains, during log and late-log phases of growth (Fig. 5A).Under (NH 4 ) 2 SO 4 -supplemented condition, the Ox-AB strain accumulated the highest level of intracellular lipids (26.9%w/DCW) during the log phase, while Ox-A had the highest content of intracellular lipids (29.6%w/DCW) during late-log phase of growth.Late-log phase of growth was primarily prominent for higher secretion of FFAs than log phase, while (NH 4 ) 2 SO 4 supplementation synergistically increased the secretion of FFAs, in particular the Ox-A strain (17.5%w/DCW) (Fig. 5B).The Ox strains in this study had higher total amounts of intracellular lipids and extracellular FFAs at the log phase of cell growth, as presented in Fig. 5C, while at the late-log phase, only Ox-A strain contained the highest total amount of them at about 47.2%w/DCW (Fig. 5C).
The other linkage metabolites related to carbon storage in cyanobacteria, including glycogen and PHB accumulation, were also determined (Fig. 6).The least glycogen content was found in the Ox-A strain at the log phase of cell growth under both growth conditions, while the higher accumulation of glycogen was induced by (NH 4 ) 2 SO 4 in the Ox-B strain at approximately 16.2 and 21.4% w/DCW at the log and late-log phases, respectively (Fig. 6A).It is important to point out that the growth phase had a relationship with the glycogen accumulation, as clearly indicated by the reduced glycogen content noticed in the late-log phase.Moreover, the (NH 4 ) 2 SO 4 supplementation could decrease glycogen accumulation in WT strain at both log and late-log phases of growth (Fig. 6A).On the other hand, polyhydroxybutyrate (PHB) accumulation, another carbon storage in cyanobacteria, was trivially induced in Ox strains that were grown in normal BG 11 and BG 110 + (NH 4 ) 2 SO 4 media (Fig. 7B).However, (NH 4 ) 2 SO 4 treatment could slightly induce PHB production in the Ox-A strain (3.85%w/DCW), but almost abolished PHB accumulation in the WT strain.Moreover, the transcript levels of genes including, amt1, aroB, phaA, accA, glgX, plsX, lipA, aas, 16s rRNA in all strains grown at late-log phase of growth under both conditions were shown in Fig. 2. The transcript amount of the amt1 gene, encoding the AMT transporter, was normally expressed at a high level in WT and higher in all Ox strains.In contrast, the aroB transcript levels, encoding 3-dehydroquinate synthase in 3-dehydroquinate (DHQ) synthesis, were lower in WT and Ox-A strains under both BG 11 and BG 110 + (NH 4 ) 2 SO 4 conditions.It is interesting to note that the glgX gene transcript kept track of the gene involved in the breakdown of glycogen.High levels of glgX transcript were expressed regularly in both WT and Ox strains, and the (NH 4 ) 2 SO 4 treatment only slightly lowered glgX transcript levels in Ox strains relative to WT.On the other hand, in carbon flux via the acetyl-CoA route, the accA gene transcript, encoding acetyl-CoA carboxylase subunit A, was highly accumulated during the late-log phase in all strains, except for the Ox-A strain, which had a lower amount of accA transcript under the (NH 4 ) 2 SO 4 condition.Additionally, under typical BG 11 condition, the WT strain had higher level of plsX gene transcript expression than OX strains, which is connected to the synthesis of membrane lipids.The (NH 4 ) 2 SO 4 treatment had an influence on lower levels of plsX in all strains, while the highest amount was found in the Ox-A strain.Moreover, it is crucial to monitor membrane lipid degradation; high amounts of lipA transcript, encoding lipase A enzyme, were regularly expressed in all strains under normal BG 11 condition, with Ox-A accumulating its lowest level.Unexpectedly, treatment with (NH 4 ) 2 SO 4 significantly caused an elevation of the lipA transcript level in the Ox-A strain (Fig. 2A,B).The recycling reaction of free fatty acid (FFA) products from membrane lipid degradation was also determined by aas transcript level, encoding acyl-ACP synthetase.Despite the Ox-A strain having the lowest level, both WT and Ox strains consistently accumulated a high level of aas transcript under both BG 11 and BG 110 + (NH 4 ) 2 SO 4 conditions.For another route from acetyl-CoA to PHB synthesis, the amount of phaA transcript engaged in PHB synthesis in Ox-B strain was expressed in a manner comparable to that of the WT, while lower levels of phaA transcript were found in the Ox-A and Ox-AB strains under the normal BG 11 condition.The (NH 4 ) 2 SO 4 treatment resulted in an upregulated phaA transcript level in the Ox-A strain, which was consistent with a higher induction of PHB under the same condition.

DPPH radical scavenging activity and components of mycosporine-like amino acids (MAAs) under ammonium sulphate supplementation
The DPPH radical scavenging capacity of methanol cell extracts from all strains was first assessed (Fig. 7A), and then pigment-free extracts were examined for DPPH radical scavenging activity and MAA component identified by HPLC (Fig. 7B,C).Results pointed out that late-log phase of growth had an impact on higher DPPH radical scavenging activity (73-78%) in cell extracts from all strains than log phase did (Fig. 7A).The (NH 4 ) 2 SO 4 treatment did not severely lower the ability of cell extracts to scavenge DPPH radicals.Although the pigment-free extracts had lower DPPH scavenging activity than methanol cell extracts by about 2-8 folds, it is worthwhile to note that at the late-log phase of cell growth, pigment-free extracts from Ox strains, which considerably included MAAs, had stronger DPPH scavenging activity than WT (Fig. 7B).
In Fig. 7C, the HPLC chromatogram, at both 334 and 310 nm, peak no. 4 (or P4) of methanolic extract at the retention time of 4.6 min was a major component of MAAs in WT under normal BG 11 condition during late-log phase of cell growth, whereas P1, P2, P3, and P5 contributed only a minor fraction.Under typical BG 11 condition, Ox strains, particularly Ox-A and Ox-AB, were shown to contain higher percentage of P4 and increased fraction of P1.Remarkably, all strains appeared to have the largest component of P4 exerted by the (NH 4 ) 2 SO 4 treatment, more than 80%, with the exception of the Ox-A strain detected by HPLC at 334 nm, which had the highest proportion of P1 (59%) instead.Furthermore, Ox-AB had a P2 component that had been inducibly increased by (NH 4 ) 2 SO 4 treatment up to 4%.
The fold change of transcripts and products under the late-log phase of growth between OX and WT is presented in Fig. 8. Ammonium supplementation had an impact on the increased fold change of PHB accumulation in OX strains compared to WT, although the OX strain preferred to accumulate glycogen content rather than PHB (Figs. 6 and 8).When compared to WT, OX strains appeared to have a lower fold change in the glgX transcript level, which is implicated in the breakdown of glycogen.On the other hand, increased antioxidant activity was found in OX strains, with a higher fold change in comparison with WT.

Discussion
In this study, we metabolically constructed the engineered Synechocystis sp.PCC 6803 strains by overexpressing amt1 and aroB genes related to ammonium transporter (AMT) and 3-dehydroquinate (DHQ) synthesis, an upstream to aromatic amino acid and mycosporine-like amino acid syntheses, respectively.Three engineered strains, including the amt1-overexpressing strain (Ox-A), the aroB-overexpressing strain (Ox-B), and the double amt1_aroB-overexpressing strain (Ox-AB), had different metabolic flows exerted by ammonium sulphate ((NH 4 ) 2 SO 4 ) treatment compared with normal BG 11 containing NaNO 3 as a major nitrogen source 5,6,35 .
Lower growth rates of all strains were caused by (NH 4 ) 2 SO 4 supplementation when compared to normal BG 11 condition (Fig. 3).According to previous findings, the PSII was found to be disrupted by the higher concentration of NH 4 + in the medium, which increased the toxicity to the cell in cyanobacteria, algae, and plants [36][37][38][39][40] .Additionally, gene overexpression with interruption of the psbA2 gene by double homologous recombination contributed to the reduced growth of the Ox strains, including Ox-A and Ox-B.Regarding ammonium sulfate supplementation, all psbA genes (psbA1, psbA2, psbA3) encoding D1 protein in the PSII were indicated to contribute to ammonium tolerance in Synechocystis sp.PCC 6803 41 .However, the Ox-A and Ox-AB strains with amt1 gene overexpression grew better with higher growth rate than other strains (Fig. 3).We also demonstrated that the intracellular pigment contents of Ox strains, including chlorophyll a and carotenoids, were slightly different and lower than in WT while growing in normal BG 11 containing NaNO 3 .However, the aroB-overexpressing strain (Ox-B) strain, which had the lowest pigment contents, was especially affected by the replacement of (NH 4 ) 2 SO4 in the medium (Fig. 3C-F).We thoughtfully speculated that aroB overexpression might promote pyruvate conversion to PEP and DAHP, respectively, a crucial substrate for DHQ synthesis by DHQ synthetase (encoded by aroB gene) (Fig. 8).This would result in lower flow direction from pyruvate to carotenoid and chlorophyll biosynthesis.There was evident support that the Ox-B cells, growing in BG 110 + (NH 4 ) 2 SO 4 during the first 4 days of cultivation (Fig. 4), showed an apparently light green culture in accordance with their lower OD 730 and pigment contents.
For carbon flow-directed products, we showed that Synechocystis sp.PCC 6803 WT cells regularly utilized NaNO 3 in typical BG 11 growth medium for intracellular lipids and stored glycogen as a major carbon storage with a trace quantity of polyhydroxybutyrate (PHB) (Figs. 5 and 6).While WT cells maintained their intracellular lipid levels in the log and late-log phases of growth, they secreted more free fatty acids (FFAs) in the latelog phase.The amt1 and/or aroB overexpression in Synechocystis sp.PCC 6803 showed a significant increase of intracellular lipid levels under normal BG 11 condition (Fig. 5).Our results indicated that amt1 overexpression in Ox-A, involved in ammonium transporter, dramatically increased the carbon flux required to synthesize intracellular lipids (29.6%w/DCW) and extracellular FFAs (17.5%w/DCW) under the influence of (NH 4 ) 2 SO 4 treatment, in particular, during the late-log phase of growth (Fig. 8).This finding was also supported by the increased fold changes of plsX, related to membrane lipid synthesis, and lipA, related in FFA production from membrane degradation, at transcript levels of about 1.13 and 1.92, respectively, when compared with those of WT.It is worth noting that when lipA transcript level was upregulated, this would indicate that intracellular FFA levels were also elevated to an excessive level, which could cause cell damage by disrupting the electron transport chain and destabilizing proteins located on thylakoid membranes 28,[42][43][44] .Therefore, in order to lessen FFA toxicity, cells have the ability to recycle FFAs into fatty acyl-ACP or secret FFA out of cells.Additionally, the Ox-A strain favored the production of lipids while accumulating the least amount of glycogen in order to balance the metabolism and storage of carbon (Fig. 6).In contrast, the aroB overexpression in Ox-B considerably contributed to DHQ synthesis, preferred to retain a high amount of glycogen (5.72 times more than WT), and kept lipids levels steady under the (NH 4 ) 2 SO 4 condition (Figs. 6 and 8).It is in line with results of glgX transcript amounts related to glycogen breakdown, which demonstrated a lower level in Ox-B strain when compared to WT, with a 0.81 fold difference (Fig. 8).Moreover, we also demonstrated that at the late-log phase of growth, all Ox strains had more accumulations of PHB than the WT strain, albeit having less than 5%w/DCW.Despite the fact that nitrogen deprivation is known to cause a significant increase in PHB 45,46 , we suggested that ammonium treatment may have had a minor impact on the ability of the amt1-overexpressing strain (Ox-A) to produce more PHB during the late-log phase of growth.
On the other hand, we demonstrated that the component of mycosporine-like amino acids (MAAs) component of nitrogen flow-directed products was partially responsible for the DPPH radical scavenging activity (Fig. 7).Under ammonium treatment, methanol extracts from all Ox strains, especially Ox-B and Ox-AB strains, had a notable increase in DPPH radical scavenging activity.Results suggested that the aroB overexpression contributing to the synthesis of DHQ, a crucial intermediate in the biosynthetic pathways for aromatic amino acids and MAAs, increased the capacity of antioxidant activity.It is also crucial to take into account that the late-log growth stage significantly raised DPPH radical scavenging activity and caused a greater fold increase than WT (Fig. 8).On the other hand, by using the GS/GOGAT cycle of glutamate and glutamine synthesis, amt1 overexpression, which encodes ammonium permease in the ammonium transporter, induced a certain improvement in the intracellular nitrogen pool 16,47 .According to previous studies, after 24-48 h of culture of Synechocystis cells under the NH 4 Cl condition, numerous amino acids were enhanced in comparison to the NaNO 3 condition due to the different mechanism of nitrogen assimilation in the GS/GOGAT cycle between these two conditions 48,49 .In recent study, the aroB gene overexpression in Escherichia coli markedly enhanced aromatic amino acid (AAA) production 50 .
In cyanobacteria and algae, mycosporine-like amino acids (MAAs) have been predicted to synthesize via the first part of the shikimate pathway, where 3-dehydroquinate (DHQ) acts as a precursor for MAAs via gadusols 51,52 .MAAs in microalgae play a role in protective actions for survival against UV radiation, salinity, and other environmental challenges 53 .Mycosporine-glycine is the major type of MAAs, and it can be transformed into secondary MAAs such as shinorine, porphyra-334, palythine-serine, and others 52,54,55 .In the methanolic extract of the cyanobacterium Anabaena doliolum, three MAAs, including mycosporine-glycine, porphyra-334, and shinorine, www.nature.com/scientificreports/were apparently identified by HPLC 56 .It is consistent with our HPLC at 334 nm results that demonstrated three distinct peaks of MAAs, including P4, P2, and P1, with retention times of 4.6, 2.9, and 2.1 min, respectively, and a small peak of P3 at a retention time at 3.9 min (Fig. 7).However, when detected by HPLC at 310 nm, the main P4 was dominant under both conditions (Fig. 7C).Prior research has confirmed that UV exposure caused the production of MAAs in Synechocystis sp.PCC6803, including mycosporine-taurine (M-tau) 31,57 , dehydroxxylusujirene 31,57 , M-343 57 , and mycosporine glycine 58 .The P1 to P5 fractions yielded similar findings to HPLC data of methanolic MAAs extract from a cyanobacterium Anabaena doliolum 56 , even though we did not identify each separated peak using HPLC with diode array detector.To pinpoint the exact MAA type, a particular extraction and detection approach for MAAs in Synechocystis sp.PCC6803 is required.Under typical BG 11 condition during late-log phase of growth, the component of MAAs in Synechocystis sp.PCC 6803 WT strain consisted mostly of P4 fraction (52%),with a small portion of P1 (8%), P2 (1%), and P3 (1%) and a substantial proportion of P5 (38%).Our finding suggested that the mycosporine-glycine component, a primary component previously identified in Anabaena doliolum 56 , was present in a comparable HPLC chromatogram of the P4 fraction at 4.6 min retention time.Despite the fact that shinorine was defined as the MAAs component of the P1 fraction (rt = 2.1 min) 56 , the Synechocystis sp.PCC6803 wild type was previously reported to lack shinorine 59 .
As in comparison with WT, the results indicated that the amt1 and/or aroB overexpression had a substantial impact on the increased major component of P4 and P1.Furthermore, it was strikingly changed on the MAAs' component induced by BG 110 + (NH 4 ) 2 SO 4 medium.When ammonium was used as the nitrogen source, the P4 composition in the WT strain was dramatically boosted by up to 80%.A similar increase in P4 fraction was present in Ox-B and Ox-AB, with a certain composition of about 87% and 88%, respectively.In addition, the P2 component was apparently increased in Ox-AB by up to 4% in this condition, detected by HPLC at 334 nm.
In contrast, the prominent component in Ox-A shone a high light on P1 fraction, up to 59%, as a result of ammonium treatment.Our finding thus revealed that the strategy by which Synechocystis sp.PCC 6803 utilized distinct nitrogen sources, in this case NaNO 3 and (NH 4 ) 2 SO 4 , had an impact on the component of MAAs that was connected to its antioxidant ability.Our findings demonstrated that Ox-AB, which had higher compositions of P4 and P2 than WT, had the best capacity for DPPH radical scavenging due to the combined overexpression of amt1 and aroB genes.In addition, it was previously discovered that mycosporine-2-glycine (M2G), which is more active than other MAAs such as shinorine, porphyra-334, and palythine, efficiently increased antiinflammatory and antioxidant properties by blocking the formation of advanced glycation end-products (AGEs) in lipopolysaccharide-stimulated RAW 264.7 macrophages 60 .Furthermore, increasing the nitrate and phosphate content in the medium proved another effective way to promote MAAs synthesis by Fischerella sp.F5 61 .

Strains and culture conditions
The host propagation, Escherichia coli DH5α strain was grown either on an agar plate or in a liquid medium of Luria Bertani (LB) containing 35 µg/mL of chloramphenicol (Cm) at 37 °C.Cyanobacterium Synechocystis sp.PCC 6803 wild type (WT) was derived from the Berkeley strain 6803 isolated from fresh water in California, USA 62 .Synechocystis sp.PCC 6803 strain was cultivated in normal BG 11 medium 35 using a rotary shaker at 28 °C and continuous light illumination of 50 µmol photons m -2 s -1 .All engineered strains in this study, including Ox-A, Ox-B, and Ox-AB (Table 1) were cultured in a normal BG 11 medium containing 35 µg/mL of chloramphenicol at the same growth condition.

Constructions of recombinant plasmids
To construct the recombinant pECm_amt1, pECm_aroB, and pECm_amt1/aroB plasmids (Table 1), the pEERM vector containing the the chloramphenicol resistance cassette gene (Cm r ) was used as a model vector for cloning and expressing genes 63 .The sequences of amt1 (sll0108) and aroB (slr2130) genes were retrieved from the Cyanobase database.The amt1 (sll0108) and aroB (slr2130) gene fragments with sizes of 1548 and 1398 bp were amplified by PCR using a pair of sll0108_F and sll0108_R primers, and another pair of slr2130_F and slr2130_R primers, respectively (Supplementary Information Table S1).The recombinant pECm_amt1 plasmid was constructed by inserting a homologous amt1 gene fragment into the pEERM vector between the XbaI and BcuI restriction sites.Additionally, the recombinant pECm_aroB plasmid was created by inserting an aroB gene fragment into the pEERM vector between the BcuI and PstI restriction sites.Ultimately, the recombinant pECm_amt1/aroB plasmid was constructed by introducing the aroB fragment into the recombinant pECm_amtI plasmid between the BcuI and PstI restriction sites.

Figure 1 .
Figure 1.Genomic maps of the engineered Synechocystis strains, including Ox-A (A, B), Ox-B (A, C), and Ox-AB (A, D), respectively.The specific pairs of primers (Supplementary Information TableS1) were used to confirm the complete integration of all transformants by PCR analysis.For the double homologous recombination (A), the amt1 or aroB gene recombination occurred between the conserved sequences of psbA2 gene in Synechocystis sp.PCC 6803 wild type (WT).For Ox-A strain (B), PCR products using UUSpsbA2 and DDSpsbA2 primers, Lane M: GeneRuler DNA ladder (Fermentas Life Sciences, MD, USA); Lane 1: negative control using WT as template, Lanes 2-6: Clones No. 1-5.Only positive clones no. 3 and 4 in respective Lanes 4 and 5, were obtained.For Ox-B strain (C), PCR products using UUSpsbA2 and DDSpsbA2 primers, Lane M: GeneRuler DNA ladder, Lane 1: negative control using WT as template; Lanes 2-6: Clones No. 1-5.Only positive Clones No. 3-5 in respective Lanes 4-6, were obtained.For Ox-AB strain (D), the single homologous recombination was confirmed by PCR using various pairs of primers (Supplementary Information TableS1), Lane M: GeneRuler DNA ladder; Lanes 1 and 2: negative control using WT as template and a transformant, respectively, with UUSll0108_F and Cm_R primers; Lanes 3 and 4: negative control using WT as template and a transformant, respectively, with Cm_F and pEbb_R primers, Lanes 5 and 6: negative control using WT as template and a transformant, respectively, with pEbb_F and Slr2130_R primers, Lanes 7 and 8: negative control using WT as template and a transformant, respectively, with Slr2130_F and pEbb_R primers.

Figure 2 .
Figure 2. The transcript levels (A) and relative transcript intensity ratios (B) of the amt1, aroB, phaA, accA, glgX, plsX, lipA, aas, and 16s rRNA performed by RT-PCR in Synechocystis WT, Ox-A, Ox-B, and Ox-AB strains.Cells were grown in normal BG 11 and BG 110 + (NH 4 ) 2 SO 4 media and analyzed at day 10 of cultivation (late-log phase).In (B), the error bars represent standard deviations of means (mean ± S.D., n = 3).The statistical difference of the results between the values of WT and engineered strain is represented by an asterisk, *P < 0.05.All cropped gels were taken from the original images of RT-PCR products on agarose gels as shown in Supplementary Information Figures S1 and S2.

Figure 3 .
Figure 3. Growth (A, B), chlorophyll a (C, D) and carotenoid (E, F) contents, and oxygen evolution rates (G, H) of Synechocystis WT, Ox-A, Ox-B, and Ox-AB strains cultured under normal BG 11 medium (while background; A, C, F, G) and BG 110 medium with (NH 4 ) 2 SO 4 supplementation (BG 110 + (NH 4 ) 2 SO 4 , gray background; B, D, F, H) during 16 day of cultivation.The oxygen evolution rates (E, F) of all strains were determined from cell culture growing at the log (day 6), late-log (day 10), and early-stationary (day 12) phases.The error bars represent standard deviations of means (mean ± S.D., n = 3) with the statistical difference of the results between WT and engineered strain represented by an asterisk, *P < 0.05.

Figure 4 .
Figure 4. Images of cell cultured flasks of Synechocystis WT, Ox-A, Ox-B, and Ox-AB strains, grown in normal BG 11 and BG 110 + (NH 4 ) 2 SO 4 media during 16 days of cultivation.

Figure 5 .
Figure 5. Contents (%w/DCW) of intracellular lipids (A), extracellular free fatty acids (FFAs) (B), and total contents of intracellular lipids and extracellular FFAs (C) in Synechocystis WT, Ox-A, Ox-B, and Ox-AB strains.Cells were grown in normal BG 11 (white background) and BG 110 + (NH 4 ) 2 SO 4 (gray background) media and analyzed at day 6 and 10 of cultivation represented cells at log phase and late-log phases of cell growth, respectively.The error bars represent standard deviations of means (mean ± S.D., n = 3).The statistical difference of the results between WT and engineered strain was represented by an asterisk, *P < 0.05.

Table 1 .
Strains and plasmids used in this study.P psbA2 strong psbA2 promoter, cm r chloramphenicol resistance cassette.